Antigen receptor genes are assembled from their component gene segments by a series of site-specific DNA recombination reactions known as V(D)J recombination. Gene segments which undergo this reaction are flanked by conserved DNA elements called recombination signal sequences (RSSs). These elements are recognized in a pairwise fashion by the lymphoid-specific proteins, RAG1 and RAG2, which introduce a pair of double-strand DNA breaks immediately adjacent to the RSSs. The four resultant DNA ends are then joined to form a signal joint and a coding joint. These joining steps utilize components of the dsDNA break repair machinery expressed in all cells. Seven complex genetic loci undergo V(D)J recombination including the immunoglobulin (Ig) mu, kappa, and lambda loci and the T cell receptor (TCR) alpha, beta, gamma, and delta loci. The rearrangement of these loci is regulated in several ways: a) Ig genes fully rearrange only in the B lineage and TCR genes only in the T lineage; b) within each lineage, antigen receptor gene rearrangement is highly ordered, with Ig mu and TCR beta rearrangement preceding Ig kappa and TCR alpha rearrangement for example; and c) an individual B or T cell makes only one productive (in frame) rearrangement at a given locus (allelic exclusion). The aim of this research proposal is to understand how a common V(D)J recombinase recognizing a conserved RSS can generate an exquisitely regulated pattern of gene-segment rearrangement. A wealth of correlative data has led to the hypothesis that accessibility of rearranging gene segments in chromatin determines the targeting of the V(D)J recombinase. The investigator showed recently that recombinant RAG1 and RAG2 when supplemented with nuclear extract could recognize and cleave RSSs in vitro within nuclei from RAG-deficient lymphoid cells. The pattern of cleavage corresponds to the state of development of the nuclei, mimicking the normal pattern of regulation of the recombinase. He went on to show that precisely positioned mononucleosomes can prevent RAG cleavage and that transcriptional enhancers within rearranging loci were critical for accessibility and function of the recombinase. In addition, it was found that nuclear proteins in addition to RAG1 and RAG2 were required for the recombinase to recognize and cleave RSSs within purified genomic DNA substrates. Experiments are proposed to further examine the role of nucleosomes in regulating V(D)J recombination, to purify and molecularly clone factors which help target recombination, to ask whether the V(D)J recombinase has transposase activity in vivo, and to determine the ability of the catalytic domain of RAG2 to complement lymphoid development in a RAG2 mutant mouse.